Making competent cells

Be sure to keep everything cold!!

Items needed:

100mL LB in flask
150 mL cold H2O
3mL cold 10% glycerol
lots of ependorphs
centrifuge tubes
ice
dry ice



Protocol:

Innoculate 100mL of a LB with 1mL fresh overnight
Grow cells with vigorous shaking to OD 600 of 0.5 to 1 - about 4 hours
Chill the culture on ice for 15 to 30 min, centrifuge in a cold rotor at 4000X for 15 min
Decant supernatant
Resuspend pellets in total of 100mL of cold water and centrifuge
Decant supernatant
Resuspend in 50mL of cold water and centrifuge
Decant supernatant
Resuspend in 2mL 10% cold gycerol and centrifuge
Decant supernatant
Resuspend in a final volume of .3 mL in 10% cold glycerol - cell conc should be about 3^10 cells/mL
Aliquot cells into 40microL amounts and freeze on dry ice.  Store at -80 deg C
Cells are good for at least 6 months